Sequestration of ass in the periphery in the absence of immunomodulating agent as a therapeutic approach for the treatment or prevention of beta-amyloid related diseases

ABSTRACT

The present invention describes a method of administering an Aβ-binding agent or drug which has affinity for amyloid beta (Aβ) in the periphery (blood) and reducing Aβ levels in the brain without the need for the agent or drug to enter the brain itself. The Aβ-binding agents utilized in the methods of the invention are preferably non-immunomodulating agents (e.g., antigenic peptides or antibodies) and bind to Aβ in the periphery, or blood. Such compounds do not significantly cross the blood/brain barrier, and yet they lower amyloid (Aβ) levels in the brain, thereby serving as safer, therapeutic and prophylactic treatments against diseases associated with Aβ in the brain, e.g., Alzheimer&#39;s Disease and amyloid angiopathy, as well as against other AD-related amyloidoses.

The work described herein is supported in part by a grant from theNational Institutes of Health, National Institute of Aging, AG17585.

FIELD OF THE INVENTION

The present invention relates to improved drug delivery methods and thediscovery and development of novel compounds and drugs for the treatmentand prevention of neurological diseases and disorders associated withβ-amyloid, such as Alzheimer's disease, β-amyloid related problems inDown's syndrome and vascular dementia (cerebral amyloid angiopathy) andother amyloidosis diseases. The invention further relates to diagnosticand screening methods for determining or identifying the aforementioneddiseases and disorders associated with β-amyloid in patients.

BACKGROUND OF THE INVENTION

Alzheimer's Disease (AD) is the most common cause of chronic dementia,with approximately two million people in the United States having thedisease. The histopathologic lesions of Alzheimer's disease (i.e.,neuritic amyloid plaques, neurofibrillary degeneration, andgranulovascular neuronal degeneration) are found in the brains ofelderly people with Alzheimer's dementia.

It is estimated that ten percent of individuals older than 65 years ofage have mild to severe dementia. The number of such lesions correlateswith the degree of intellectual deterioration. This high prevalence,combined with the rate of growth of the elderly segment of thepopulation, make dementia (and particularly AD) one of the mostimportant of the present-day public health concerns.

An invariant feature of Alzheimer's disease (and AD in Down's syndrome)is the deposition of the small, i.e., approximately 40 to 42 residues,amyloid beta (also referred to as Aβ or Abeta herein) peptide asinsoluble β-amyloid plaque in the brain parenchyma. (G. G. Glenner etal., 1984, Appl. Pathol., 2(6):357-69; G. G. Glenner et al., 1984,Biochem Biophys Res Commun., 120(3):885-90; G. G. Glenner et al., 1984,Biochem Biophys Res Commun., 122(3):1131-5). In cerebral amyloidangiopathy, Aβ is deposited in the vasculature. Aβ is generated byproteolysis of the approximately 100 kDa amyloid precursor protein(APP), a broadly expressed type-1 transmembrane protein that is foundprimarily in the trans Golgi network (TGN) and at the cell surface(reviewed in B. De Strooper and W. Annaert, 2000, “Proteolyticprocessing and cell biological functions of the amyloid precursorprotein.” J. Cell. Sci., 113(Pt 11)(7):1857-1870). The β-amyloidprecursor protein APP is further described in D. J. Selkoe et al., 1988,Proc. Natl. Acad. Sci. USA., 85(19):7341-7345; R. E. Tanzi et al., 1988,Nature, 331(6156):528-530; and E. Levy et al., 1990, Science,248(4959):1124-1126.

Amyloid plaques containing Abeta (Aβ) peptides are one of the mostsignificant pathological features of the human Alzheimer's diseasebrain. Drugs that reduce brain Aβ levels, or remove plaques, areconsidered to be the most likely to be effective in the treatment orprevention of AD. To date, treatments for AD have focused on the use ofanti-Aβ antibodies or peptides which evoke the production of anti-Aβantibodies, i.e., vaccine therapy. (see, for example, D. Schenk et al.,1999, Nature, 400(6740):173-177 and F. Bard et al., 2000, Nat Med,6(8):916-919).

The presumed mode of action for such antibody, or immunomodulatory,treatments is in the clearance of Aβ directly from the brain due to theentry of antibodies into the brain. A side effect of vaccination is anincrease of peripheral Aβ levels as levels of Aβ decrease in the brain,thus resulting in a little understood change in the dynamics between thetwo systems, brain and periphery.

Vaccination involving anti-Aβ antibodies is a potentially ineffectiveand possibly even dangerous approach for treatment of AD patients,particularly the elderly who lack (or have less) immune responsiveness,due to the risk of provoking autoimmune diseases.

Clearance of Aβ from the brain has been reported using immunization ofAβ peptides or passive immunization with anti-Aβ antibodies (D. Schenket al., 1999, Nature, 400(6740):173-177 and F. Bard et al., 2000, NatureMed., 6(8):916-919). The proposed mechanism for this clearance ismicroglial phagocytosis carried out by brain immune cells, i.e.,microglia, that have been activated by elicited or injected anti-Aβantibodies.

A goal in the field of therapy and prevention of AD and amyloid-relateddiseases is the discovery and development of new drugs that areeffective due to their mode of action in the periphery, rather than inthe brain, thus obviating the need to enter the brain itself andovercoming the problems encountered in efficient dosage andeffectiveness due to the blood/brain barrier. The present inventionsatisfies this goal by providing new methods of treatment and preventionfor AD and other amyloid-related diseases, and by describing new typesof drugs and compounds that serve to treat or prevent disease via theblood.

SUMMARY OF THE INVENTION

The present invention provides methods and compounds (also termed drugs,substances, reagents, or agents, preferably bioactive agents) employedtherein for sequestering Aβ in the blood, or blood components, such asplasma, i.e., the periphery, thereby reducing Aβ levels in the brain fortreatment or prevention of beta-amyloid related diseases. The compoundsof the invention have an affinity for (i.e., “sequester”) Aβ, and bindto and sequester Aβ in the blood, or periphery, e.g., plasma, withoutneeding to enter the brain itself. According to this invention, suchcompounds do not (and do not need to) cross the blood/brain barrier, andyet they significantly lower amyloid (Aβ) levels in the brain. Suchcompounds have been shown in animal models of disease, e.g., atransgenic AD mouse model, to lower Aβ levels in the brain bysequestering Aβ in the periphery, e.g., plasma. That the inventionprovides a method and drugs used therein which obviate the need for adrug to enter the brain itself, while still significantly loweringamyloid (Aβ) levels in the brain, offers a great improvement over drugsthat currently must enter the brain to have an effect on Aβ levels inthe brain.

Thus, it is also an aspect of the present invention to provide a methodof treating or preventing AD comprising administering to an individualin need thereof a compound or drug having an affinity for Aβ, whichbinds to Aβ in the periphery, wherein such a compound or drug,preferably a non-immune related compound or drug, and also preferably,an agent other than an antibody or an immunomodulating agent, sequestersAβ in the periphery and leads to a reduction in Aβ levels in the brain.For delivery to the periphery, such Aβ-binding compounds are preferablyintroduced intravenously or subcutaneously; however, any method ofintroducing the compound into the blood stream (including via pumps) isacceptable and suitable in accordance with this invention.

It is another aspect of the present invention to provide a method ofreducing amyloid (or soluble/insoluble Aβ) levels in the brain of apatient undergoing treatment by obviating the need to introduce anAβ-binding drug or compound directly, or indirectly, into the brain.According to the invention, the effectiveness of the method in which theAβ-binding drug sequesters Aβ in the bloodstream and removes it from thebrain is at least as high as a vaccine approach involving the productionof antibodies that cross the blood/brain barrier, enter into the brain,and act in the brain.

It is yet a further aspect of the present invention to provide a methodfor diagnosing, screening, or monitoring treatment of diseases involvingβ-amyloid or amyloidoses, such as AD, comprising determining anelevation of Aβ levels in the periphery, e.g., plasma, particularly at atime, or series of times, following treatment. In accordance with thisaspect of the invention, the elevation of Aβ levels in the periphery,e.g., plasma, serves as a diagnostic marker of diseases involvingβ-amyloid, particularly, AD.

Further aspects, features and advantages of the present invention willbe apparent when considered in connection with the further disclosure ofthe invention hereinbelow.

DESCRIPTION OF THE INVENTION

In one of its aspects, the present invention describes compounds (drugs)which have an affinity for, i.e., “sequester”, Aβ in the blood, or bloodcomponents, e.g., plasma, (periphery) and which reduce Aβ levels in thebrain without the need of the compounds (e.g., drugs or bioactiveagents) to enter the brain itself. Such compounds sequester Aβ in theperiphery and alter the periphery/brain dynamics so as to reduce Aβ inthe brain by virtue of their effective sequestration of Aβ in theperiphery.

According to this invention, such compounds are preferably brainimpermeable and essentially do not (and do not need to) cross theblood/brain barrier following administration or introduction into arecipient, and yet they significantly lower amyloid (Aβ) levels in thebrain. Also in accordance with this invention, such compounds have beenshown in animal models of disease, e.g., AD mouse models, to lower Aβlevels in the brain by sequestering Aβ in the periphery, e.g., plasma.(Example 1). It is thus an aspect of the invention that the Aβ-bindingagent, drug, compound, and the like, effectively sequesters Aβ in theperiphery following administration in the periphery. Preferably, greaterthan about 50% of the Aβ-binding agent, drug or compound remains in theperiphery versus the brain following administration in the periphery.More preferably, about 90% or more of the Aβ-binding agent, drug orcompound remains in the periphery versus the brain followingadministration in the periphery

In addition, the finding of elevated Aβ in the periphery, particularlyin plasma, preferably in conjunction with the administration of agentsthat bind Aβ and sequester Aβ in the periphery, can serve as adiagnostic marker of β-amyloid-related diseases, especially, AD. Theelevation of levels of Aβ in the periphery can further serve as a meansof monitoring the effectiveness of treatment of a disease involving Aβ,particularly with a drug or agent that binds and sequesters Aβ in theperiphery, thereby leading to its elevation in the periphery. Accordingto this embodiment of the invention, an elevation of Aβ in the peripheryreflects an amount of Aβ that is increased relative to that found innormal individuals, such as in plasma, or a base level of Aβ, e.g., inplasma, in individuals who serve as controls. Determining and/ormeasuring levels of Aβ can be performed using routine techniques asknown in the art, such as radioimmunoassays (RIAs), nonradioactiveimmunoassays, such as enzyme linked immunoassays (ELISAs), westernblotting, dot blotting, mass spectrometry, etc.

Without wishing to be bound by theory, it is proposed that microglialphagocytosis is not necessary for Aβ clearance from the brain inaccordance with the present invention. Instead, sequestration in theblood, or blood component, such as plasma, i.e., the periphery, in theabsence of an immune modulating agent, by suitable Aβ-binding compoundsthat are not Aβ peptides or their derivative antibodies, serves toreduce Aβ levels in the brain and to alter the central nervous system(CNS)/periphery dynamics leading to reduction of Aβ in the brain. Asused herein, the terms immune modulating agent, and immune relatedagent, refer to an anti-Aβ antibody or a peptide against some region ofAbeta or APP that evokes the production of antibodies, which recognizean Abeta region or APP.

As a consequence of the sequestration of Aβ in the periphery accordingto this invention, higher levels of Aβ are predicted to be found in theblood (e.g., plasma)/periphery), (see Example 1), because Aβ issequestered in the blood/periphery by the Aβ-binding compounds. It isalso to be understood that the removal of bound Aβ/binding agents bycellular clearance mechanisms may effectively reduce the levels ofperipheral Aβ seen following administration of the sequestering agent.The important effect of the methods and reagents of the presentinvention is that the levels of Aβ found in the brain as a result ofkeeping Aβ sequestered in the periphery are reduced, which isadvantageous for the therapeutic effect of the method and compounds ofthe present invention.

The invention also allows for determining or monitoring a drug'seffectiveness by monitoring Aβ levels in the periphery, such as in theplasma, instead of, or in addition to, brain Aβ levels. (see, e.g.,Example 1). Methods of monitoring Aβ levels in an individual undergoingdrug treatment or therapy for amyloid related diseases involvedetermining the levels of Aβ in the individual's peripheral body fluidsample, e.g., plasma, at one or more time intervals following treatmentor therapy involving an Aβ binding agent that sequesters Aβ in theperiphery. For example, an individual can be monitored at about 1-25hours, preferably at about 2-10 hours following administration of the Aβbinding agent, or at varying time intervals therebetween, to determineif Aβ levels are elevated. Such monitoring methods are particularlyuseful for determining if a given drug treatment is beneficial, or todetermine if doses of a drug or a drug combination should be modified oradjusted during the course of treatment. In such methods, it ispreferred to use the individual's pretreatment levels of Aβ in theperiphery to compare and assess treatment and post-treatment levels ofAβ in the periphery. Controls can also include peripheral levels of Aβin disease-free (or dementia-free) individuals, as well as peripherallevels of Aβ in Individuals having an amyloid related disease, e.g., AD.Human plasma and cerebrospinal fluid levels of amyloid beta proteins,particularly, Aβ40 and Aβ42, have been reported (see, e.g., Mehta etal., 2000, Arch. Neurol., 57:100-105).

According to this invention, Aβ levels can be measured in anindividual's body fluid sample, such as blood, serum, or plasma, usingconventionally known assays that detect Aβ, for example, radioisotopicimmunoassays or non-isotopic immunoassays, e.g., fluorescentimmunoassays, chemiluminescent immunoassays and enzymatic immunoassays,such as an enzyme linked immunoassay (ELISA), as are commerciallyavailable, known and practiced in the art, for example, Beta-amyloid(Abeta) [1-40] Immunoassay (Biosource, Camarillo, Calif.; Cat. No.KHB3481); Beta-amyloid (Abeta) [1-42] Immunoassay (Biosource, Camarillo,Calif.; Cat. No. KHB3441); and Human Amyloid beta (1-40) Immunoassay(IBL, Fujioka, Gunma, Japan; Cat. No. 17713).

Typically, an ELISA assay initially involves preparing, obtaining, oremploying an antibody specific to Aβ, preferably a monoclonal antibody.In addition, a reporter antibody is used. In some ELISA protocols, thereporter antibody recognizes and binds to the anti-Aβ-specificmonoclonal antibody. To the reporter antibody is attached a detectablereagent such as a radioactive isotope, a fluorescent moiety, achemiluminescent moiety, or, in an ELISA, an enzyme, such as horseradishperoxidase or alkaline phosphatase.

As is appreciated by those skilled in the art, ELISAs can be performedin a number of assay formats. In one ELISA format, a host sample, e.g.,a patient body fluid sample, is incubated on a solid support, e.g., thewells of a microtiter plate, or a polystyrene dish, to which theproteins in the sample can bind. Any free protein binding sites on thedish are then blocked by incubating with a non-specific protein such asbovine serum albumin. The monoclonal antibody is then added to the solidsupport, e.g., the wells or the dish, and allowed to incubate. Duringthe Incubation time, the monoclonal antibodies attach to any Aβpolypeptides or peptides that have attached to the polystyrene dish.

All unbound monoclonal antibody is washed away using an appropriatebuffer solution. The reporter antibody, e.g., linked to horseradishperoxidase, is added to the support, thereby resulting in the binding ofthe reporter antibody to any monoclonal antibody which has bound to Aβpresent in the sample. Unattached reporter antibody is then washed away.Peroxidase substrate is added to the support and the amount of colordeveloped in a given time period provides a measurement of the amount ofAβ that is present in a given volume of individual or patient samplewhen compared to a standard curve.

In another ELISA format, antibody specific for a particular analyte isattached to the solid support, i.e., the wells of a microtiter plate ora polystyrene dish, and a sample containing analyte is added to thesubstrate. Detectable reporter antibodies, which bind to the analytethat has bound to the capture antibodies on the support, are then added,after the appropriate incubations and washings, and analyte-antibodycomplexes are detected and quantified.

The present invention also embraces a sandwich type ELISA immunoassaytypically performed using microliter plates. A capture antibody, thatcan be polyclonal or monoclonal, preferably a monoclonal antibody, thatspecifically recognizes an epitope in the Aβ peptide is used, along witha labeled detector antibody, e.g., an alkaline phosphatase-labeledantibody, or a horse radish peroxidase-labeled antibody, preferably amonoclonal antibody. The detector antibody also specifically recognizesan epitope in Aβ. Preferably also, the capture antibody does not inhibitbinding to Aβ. The production of both polyclonal and monoclonalantibodies, particularly monoclonal antibodies that are specific for Aβ,is performed using techniques and protocols that are conventionallyknown and practiced in the art.

In a particular embodiment according to this invention, a captureanti-Aβ antibody of the assay method is immobilized on the interiorsurface of the wells of the microtiter plate. To perform the assay, anappropriate volume of sample is incubated in the wells to allow bindingof the antigen by the capture antibody. The immobilized antigen is thenexposed to the labeled detector antibody. Addition of substrate to thewells, if the detectable label is alkaline phosphatase, for example,allows the catalysis of a chromogen, i.e., para-nitrophenylphosphate(pNPP), if the label is alkaline phosphatase, into a colored product.The intensity of the colored product is proportional to the amount of Aβthat is bound to the microtiter plate.

Standards are used to allow accurate quantitative determinations of Aβin the samples undergoing analysis. A microtiter plate readersimultaneously measures the absorbance of the colored product in thestandard and the sample wells. Correlating the absorbance values ofsamples with the standards run in parallel in the assay allows thedetermination of the levels of Aβ in the sample. Samples are assigned aquantitative value of Aβ in nanograms per milliliter (ng/ml) of blood,serum, plasma, or other body fluid.

The present invention provides a significant advantage to the treatmentand prevention of AD and amyloid-related diseases in that drugs andactive compounds according to this invention are not required to crossthe blood/brain barrier to exert their effect. Having to cross theblood/brain barrier is an enormous obstacle to developing effectivedrugs for use in the brain. This invention overcomes this obstacle. Oneof the major differences between this method and others is that it usesa non-antibody compound, or a compound that is not related to anantibody, to achieve the sequestration of Aβ, and that thissequestration has its primary effect in the periphery. A consequence ofthe method and the compounds utilized therein is a decrease in Aβ in thebrain.

In addition, a second advantage of this invention is that neither Aβpeptides, nor anti-Aβ antibodies, is administered to a host, thusnegating the risk of an adverse immune response, or the lack of aneffective immune response. For AD and amyloid angiopathy, this methodpreferably involves the use of Aβ-binding compounds and drugs of theinvention, more preferably formulated as pharmaceutically acceptablecompositions as described herein.

The method further comprises the administration of an amyloid beta(Aβ)-binding compound, agent, or drug in the periphery of an individualin need thereof, wherein the compound sequesters Aβ in the periphery andconcomitantly decreases Aβ levels in the brain of the individualundergoing treatment. According to the method of the present invention,the need to introduce an agent or drug directly into the brain, or tohave the drug cross the blood/brain barrier is obviated. Also accordingto this method, brain Aβ levels are reduced, due to the effects of theAβ-binding drugs and compounds described herein on Aβ in the periphery.

The method and Aβ-binding compounds and agents used therein inaccordance with the present invention are suitable for the treatment,both prophylactic and therapeutic, of neurological diseases anddisorders associated with β-amyloid, such as Alzheimer's disease,β-amyloid related problems in Down's syndrome and vascular dementia(cerebral amyloid angiopathy) (A. J. Rozemuller et al., 1993, Am. J.Pathol., 142(5):1449-1457) and other amyloidosis diseases. The methodinvolving peripheral sequestration of disease associated agents, e.g.,peptides, or proteins or aggregates thereof, by non-immunomodulatingagents that bind to such disease associated agents in theblood/periphery in accordance with the present invention, are alsouseful in the treatment or prevention of other cortical or vascularamyloidoses, including those caused by cystatin C (ACys), prion protein(AScr), transthyretin (ATTR), gelsolin (AGel), and Amyloid ABri (orA-WD) (see, M. Yamada, 2000, “Cerebral amyloid angiopathy: an overview”,Neuropathology, 20(1):8-22). Cortical or vascular amyloidoses are verysimilar in etiology to AD.

In accordance with the present invention, the method of deliveringAβ-binding drugs to the periphery has been shown to be at least aseffective as the vaccine approach in transgenic mouse models, if notmore so. Indeed, the use of non-toxic, non-immune related compounds anddrugs can overcome adverse immune responses that are frequentlyassociated with the use of brain-directed immunovaccines. Prior to thepresent invention, the treatment of brain amyloidosis by administeringnon-immune related Aβ-binding agents in the periphery and sequesteringor “locking away” Aβ in the blood, or periphery, has not been shown.

Suitable compounds that can be employed in the method of this inventioninclude, but are not limited to, small molecules, e.g., peptides,proteins; biologic agents; and drugs that have an affinity for Aβ andbind Aβ in the periphery. Such compounds, molecules, agents and drugshave an Aβ-binding domain that physically binds to and locks away Aβ inthe periphery. The compound, molecule, agent or drug can bind to or haveaffinity for a variety of Aβ peptides, e.g., Aβ peptides derived from AβP; Aβ peptides of different fragment lengths, e.g., Aβ40 or Aβ42, andthe like. In addition, the compound, molecule, agent or drug can bind toor have affinity for any portion of an Aβ peptide, e.g., the N- orC-terminus, or other regions of the molecule. Non-immune related and/ornon-immunomodulatory compounds or drugs are preferred. Most preferablythe compounds are nontoxic and well tolerated following their use in thetreatment and prevention methods.

An advantage of the use of molecules other than immune relatedcompounds, such as antibodies, for peripheral sequestration of Abeta(Aβ) is that non-antibody related drugs can be manipulated more easilythan antibodies. For example, sequestering compounds can be modified tobe metabolized faster by the addition of certain chemical structures, asknown and practiced in the art. For example, sequestering compounds canbe modified by the addition of side chain(s) which can modulatemetabolism. Such chemical modification of the non-antibody Aβ-bindingand sequestering compounds can improve their efficacy and reducetoxicity and/or potentially adverse side effects. The derivative ofCongo Red, an Aβ-imaging agent, as described herein, is particularlysuitable for chemical derivatization or modification.

Nonlimiting examples of such Aβ-binding compounds include compoundshaving an affinity for Aβ, particularly, cortigangliosides, such as GM1,the actin-regulating molecule gelsolin, particularly, the extracellularAβ-binding domain of gelsolin, and Aβ staining molecules, such asderivatives of Congo Red, e.g.,[1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene and 5,5′-[(1,1′biphenyl)-4,4′-diylbis(azo)]bis [2-hydroxybenzoic acid]disodium salt(chrysamine-G or CG), as described in U.S. Pat. No. 6,133,259 and WO96/34853. A preferred Aβ staining molecule is the Aβ staining dyecompound Chrysamine-G, as described in U.S. Pat. No. 6,133,259 and WO96/34853. (Example 2). Other nonlimiting examples of Aβ binding agentsthat are suitable for use in the methods of this invention include Aβimaging agents (e.g., Klunk et al., 1995, “Chrysamine-G binding toAlzheimer and control brain: autopsy study of a new amyloid probe”,Neurobiol. Aging, 16: 541-548), β-sheet breakers (e.g., Bohrmann et al.,2000, “Self-assembly of beta-amyloid 42 is retarded by small molecularligands at the stage of structural intermediates”, J. Struct. Biol.,130:232-246), β-sheet formation inhibitors (e.g., Findeis et al., 1999,“Modified-peptide inhibitors of amyloid beta-peptide polymerization”,Biochemistry, 38:6791 -6800), and the like, are encompassed for use inthe present invention.

The Aβ-binding compounds according to the invention can be incorporatedinto pharmaceutical formulations, or pharmaceutical compositions,preferably physiologically acceptable compositions, according to knownmethods, such as by admixture with a pharmaceutically acceptablecarrier, diluent, or excipient. One or more Aβ-binding compounds ordrugs comprise the pharmaceutical compositions and are formulated asactive ingredients in the compositions in a therapeutic or prophylacticamount.

The pharmaceutically, or physiologically, acceptable carrier, diluent,or excipient can be any compatible nontoxic substance suitable todeliver the compound to a host or recipient. Sterile water, alcohol,fats, waxes and inert solids may be used as carriers. In addition,pharmaceutically acceptable adjuvants, buffering agents, dispersingagents, and the like, may also be incorporated into the pharmaceuticalcompositions. The preparation of pharmaceutical compositions comprisingactive agents is well described in the scientific and medicalliterature. Examples of methods of formulation, and carriers, etc. maybe found in the latest edition of Remington's Pharmaceutical Sciences,18th Ed., 1990, Mack Publishing Co, Easton, Pa.

To formulate a pharmaceutically acceptable composition suitable foreffective administration, preferably in vivo, or even ex vivo, suchcompositions will contain an effective amount of the active compound,biomolecule, agent or drug. Pharmaceutical compositions of the presentinvention are administered to an individual in amounts effective totreat or prevent AD, amyloid angiopathy, or other Aβ-associated diseasesor conditions. The effective amount may vary according to a variety offactors, such as an individual's physical condition, weight, sex andage. Other factors include the mode and route of administration. Thesefactors are realized and understood by the skilled practitioner and areroutinely taken into account when administering a therapeutic agent toan individual.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective and sufficient amount to directly bind Aβ in the periphery,sequester it there, and reduce the Aβ levels in the brain. Thedetermination of an effective dose is well within the capability of theskilled practitioner in the art. The therapeutically effective dose canbe estimated initially either in cell culture assays, or in animalmodels, usually mice, rabbits, dogs, pigs, rats, monkeys, or guineapigs. The animal model is also used to achieve a desirable concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of the Aβ-bindingcompound or drug which ameliorates, reduces, or eliminates the symptomsor condition. In accordance with this invention, the effective dose ispreferably that which lowers, reduces, or eliminates levels of Aβ, orbuildup of Aβ, in the brain, while binding to and “locking up” Aβ in theperiphery. The exact dosage is chosen in view of the patient to betreated, the route of administration, the severity of disease, and thelike.

The concentration of the Aβ-binding drug, compound or bioactive agent inthe pharmaceutical carrier may vary, e.g., from less than about 0.1% byweight of the pharmaceutical composition to about 20% by weight, orgreater. As a nonlimiting example, a typical pharmaceutical compositionfor intramuscular administration would be formulated to contain one tofour milliliters (ml) of sterile buffered water and one microgram (μg)to one milligram (mg) of the Aβ-binding drug or compound of the presentinvention. A typical composition for intravenous infusion could beformulated to contain, for example, 100 to 500 ml of sterile bufferedwater or Ringer's solution and about 1 to 100 mg of the Aβ-binding drugor compound.

The daily dosage of the pharmaceutical, or physiologically acceptable,products may be varied over a wide range, for example, from about 0.01to 1,000 mg per adult human/per day. An effective amount of the drug isordinarily supplied at a dosage level of from about 0.0001 mg/kg toabout 100 mg/kg of body weight per day. The range is more particularlyfrom about 0.001 mg/kg to 10 mg/kg of body weight per day. Even moreparticularly, the range varies from about 0.05 to about 1 mg/kg. Ofcourse, it will be understood by the skilled practitioner that thedosage level will vary depending upon the potency or effectiveness of aparticular compound, or combination of compounds, and that certaincompounds will be more potent or effective than others.

In addition, the dosage level will vary depending upon thebioavailability of the compound. The more bioavailable and potent thecompound, the less amount of the compound will need to be administeredthrough any delivery route, including, but not limited to, oraldelivery. The dosages of the Aβ-binding compounds are adjusted, ifcombined, in order to achieve desired effects. On the other hand,dosages of the various Aβ binding agents or compounds may beindependently optimized and combined to achieve a synergistic result,wherein the pathology is reduced more than it would be if one singleagent or compound were used alone.

The pharmaceutical compositions may be provided to an individual in needof therapeutic treatment by a variety of routes, such as; for example,subcutaneous, topical, oral, intraperitoneal, intradermal, intravenous,intranasal, rectal, intramuscular, and within the pleural cavity.Administration of pharmaceutical compositions is accomplished orally orparenterally. More specifically, methods of parenteral delivery includetopical, intra-arterial, intramuscular, subcutaneous, intramedullary,intrathecal, intraventricular, intravenous, intraperitoneal, intranasaladministration, or via the pleural cavity. In addition, the compoundsaccording to the invention can be delivered via one or more routes ofadministration through the use of pumps.

Also embraced are transdermal modes of delivery, such as patches and thelike, with or without a suitable permeation enhancer. The methods andcompositions embodied by the invention provide a means by which one ormore of the Aβ-binding drugs, or medicaments, can be effectivelyadministered in a transdermal system. Frequently, compounds having poortopical absorption, or which are required at high dosage levels, aredelivered transdermally. Accordingly, a transdermal means of deliveringa drug composition (often with a permeation enhancing composition) tothe skin is that of the transdermal patch or a similar device as knownand described in the art. Examples of such devices are disclosed in U.S.Pat. Nos. 5,146,846, 5,223,262, 4,820,724, 4,379,454 and 4,956,171. Thetransdermal mode of storing and delivering the compositions onto theskin and forming the active composition is convenient and well suitedfor the purposes of the invention.

The present invention also provides suitable topical, oral, systemic andparenteral pharmaceutical formulations for use in the methods oftreatment described herein. It is to be appreciated that thecompositions containing the Aβ-binding compounds can be administered ina wide variety of therapeutic dosage forms in conventional vehicles foradministration. For example, the compounds can be administered in suchoral dosage forms as tablets or capsules (including timed release andsustained release formulations), pills, powders, granules, elixirs,tinctures, solutions, suspensions, syrups and emulsions, or byinjection. Likewise, the therapeutic compounds may also be administeredin intravenous (both bolus and infusion), intraperitoneal, subcutaneous,topical (with or without occlusion), or intramuscular form, all usingforms well known to those of ordinary skill in the pharmaceutical arts.The preferred mode of delivery for the Aβ-binding compounds according tothe present invention is intravenous.

For topical administration, the compositions of the present inventionmay be formulated in oil, water, or combinations thereof. Preferred is adermatologically acceptable formulation comprising an oil-in-wateremulsion. Examples of other dermatologically acceptable vehicleformulations of the present invention include, but are not limited to,any suitable nontoxic or pharmaceutically acceptable topical carrier,such as a solution, suspension, emulsion, lotion, ointment, cream, gel,plaster, patch, film, tape or dressing preparation, all of which arewell-known to those skilled in the art of topical skin formulations andpreparations.

The pharmaceutical compositions of the present invention can beadministered for therapeutic and/or for prophylactic purposes oftreating diseases, pathologies, or conditions related to the increase inAβ levels, or the deposition of Aβ in the brain, for example, AD andamyloid angiopathy. Prophylactic treatment is preferred, althoughtherapeutic treatment is also efficacious. For prophylacticapplications, the pharmaceutical compositions of this invention areadministered to an individual who is susceptible to, or prone to, thedisease, pathology, or condition. Such individuals can be identified bygenetic screening and/or clinical analysis, such as is described in themedical literature (see, e.g., Goate, 1991, Nature, 349:704-706 and E.H. Corder et al., 1993, Science, 261(5123):921-923). In such cases, thepharmaceutical compositions bind to or sequester AD in the periphery ata symptomatically early stage, thus preferably preventing either theinitial stages of, or the severity of, disease progression. Furthermore,prophylactic treatment can be applied to any individual wishing toundertake treatment, regardless of their susceptibility.

In therapeutic applications, the pharmaceutical compositions of thisinvention are administered to an individual in need thereof; suchindividuals already suffer from, or are thought to suffer from thedisease, pathology, or condition. In general, a dose of an Aβ-bindingcompound effective for prophylactic treatment or therapy is the same asthat for therapeutic treatment or therapy.

EXAMPLES

The following example describes specific aspects of the invention toillustrate the invention and provides a description of the presentmethods for those of skill in the art. The example should not beconstrued as limiting the invention, as the examples merely providespecific methodology useful in the understanding and practice of theinvention and its various aspects.

Example 1

Transgenic mice that develop AD-related amyloidosis, (i.e., PS/APP mice;See, L. Holcomb et al., 1998, Nature Med., 4(1):97-100) were used forthe studies described in this Example to assess how peripheralsequestration of Aβ affected brain Aβ levels. The ganglioside GM1 wasutilized as an exemplary Aβ-binding compound, since GM1 is known to bindAβ strongly, and does not appear to enter the brain. In addition, asecond compound, gelsolin, which is too large to cross the blood/brainbarrier (BBB), and is completely unrelated to GM1, but which is alsoknown to bind Aβ with great avidity, was administered peripherally toconfirm the universality of the mechanism.

PS/APP mice were injected every two days for two weeks either with GM1(number of mice=6) (15 mg/kg, ip,), with gelsolin (number of mice=3) (60μg/kg, ip), or with vehicle, (phosphate buffered saline), (number=7),into the periphery at 9 weeks of age, an age when amyloid pathology inthe brain is not visible. The mice were left for 1 week withoutinjections (the “wash-out” period) and were then sacrificed at 12 weeksof age, an age when amyloid deposition has been initiated and measurablelevels of Aβ are present in the vehicle treated controls.

The levels of Aβ in the peripheral blood were tested at three timepoints during the drug administration period (i.e., after 1 week ofinjections; after two weeks of injections; and after the washoutperiod). Data for the third time point only are shown. The levels of Aβpeptides (Aβ40 or Aβ42) in the brain and plasma were assessed by ELISAassay. All of the Aβ in the brain (including Aβ in plaques) wasextracted in 70% formic acid (FA). The levels of Aβ peptides in GM1 orgelsolin treated mice were compared with those of vehicle treatedcontrols.

The results presented in Table 1 show that there was a statisticallysignificant (p<0.05) decrease in both Aβ40 and Aββ42 in the FA-solublebrain fraction in GM1-treated mice compared with those in controlanimals. This correlates with a statistically significant increase inperipheral Aβ40 and Aβ42 at the same time point. For gelsolin, there wasalso a significant decrease in Aβ42, which correlated with astatistically significant increase in peripheral Aβ, thus confirmingthat the general principle of Aβ sequestration in the periphery beinglinked to reduction of Aβ in the CNS holds true for very different typesof compounds that have the unifying property of being able to bind Aβ inthe blood. TABLE 1 Changes of brain Abeta load [fmole/mg protein] inGM1- and Gelsolin-treated mice compared with controls (vehicle)Treatment n Abeta40 Abeta42 Vehicle 7 3630 ± 30 4100 ± 200 GM1 6 2147 ±226 2990 ± 410 % 59 73 P-value p  0.005**  0.031* Gelsolin 3 3473 ± 673 2473 ± 167 % 96 60 P-value p  0.502  0.021**results statistically significant to the 5% level (p = 0.05);**results statistically significant to the 1% level (p = 0.001).

The results presented in Table 2 show the changes in plasma load of bothAβ40 and Aβ42 in GM1-treated mice compared with control (vehicle)animals. As can be observed, there was a significant increase in plasmaAbeta levels. For GM1, both Abeta40 and Abeta42 are increased; forgelsolin, Abeta42 is increased to a greater extent than is Abeta40.Thus, the effect with gelsolin may reflect a different preference forAbeta42 over Abeta40. TABLE 2 Changes of plasma Abeta load [fmole/mlplasma] in GM1 treated mice compared with controls (vehicle) Treatment nAbeta40 Abeta42 Vehicle 7 100 ± 21 100 ± 7 GM1 6 177 ± 29 124 ± 11 p0.021** 0.036* Gelsolin 3 218 ± 87 153 ± 35 p 0.016** 0.039**results statistically significant to the 5% level (p = 0.05);**results statistically significant to the 1% level (p = 0.001).

Neither GM1 nor gelsolin is known to cross into the brain from theperiphery to any degree. In addition, as part of the studies related tothose described in this example, GM1 was introduced directly into thebrain of transgenic mice, but no change in Aβ levels was observed. Thus,the results indicate that the effects of GM1 administration in the testmice is due to the sequestration of Aβ in the periphery, thereby leadingto a change in dynamics between brain and peripheral Aβ transport. Thisis the first time that such a result has been shown for a peripherallyadministered compound that is not an antibody. As such, the inventionaffords a significant advantage to the art by describing and promotingAβ-binding compounds that require neither penetration of the brain northe evocation of an immune response, which are potentially harmful andineffective ways to modulate the risk of AD in human patients.

It will be appreciated that the use of GM1 or gelsolin in the presentexample is not limiting to the types of compounds considered to besuitable for use in the present invention. Indeed, in accordance withthis invention, any Aβ binding molecule can have the same effectfollowing peripheral administration, thus providing a powerful treatmentand therapeutic for AD sufferers, as well as those afflicted with otheramyloidoses, e.g., amyloid angiopathy.

Example 2

In this Example, as in Example 1, transgenic mice that developAD-related amyloidosis, (i.e., PS/APP mice; See, L. Holcomb et al.,1998, Nature Med., 4(1):97-100) were used to assess the Aβ-bindingcompound chrysamine G (CG) in the peripheral sequestration of Aβaccording to this invention, and to determine how the peripheralsequestration of Aβ by this compound affected brain Aβ levels. CG isknown to bind Aβ strongly, and is less brain permeable than GM1.

PS/APP mice at 10 weeks of age were injected once either with CG (numberof mice=3, dosage: 20 mg/kg) or vehicle (phosphate buffered saline,number of mice=2) into the blood stream. Blood samples were collectedprior to treatment (injection) and post-treatment at 10 minutes, 2.5hours, 5 hours and 25 hours after injection. Blood Aβ levels werecompared between pretreatment versus post-treatment at 10 minutes, 2.5,5 and 25 hours after injection. The levels of Aβ peptides (Aβ40 or Aβ42)in the plasma were assessed by ELISA immunoassay. The levels of Aβpeptides in the periphery, i.e., plasma, of CG treated mice werecompared with plasma Aβ levels in pretreatment mice at various timepoints.

Changes in plasma Aβ levels after injection with CG were compared withpretreatment plasma Aβ levels as shown in Table 3. TABLE 3 Changes inplasma Aβ levels after injection with CG Hours Aβ42 level afterinjection [% of pre-treatment time point] 2.5 110 ± 8 (p = 0.232) 5 125± 8 (p = 0.049) 7.5 125 ± 7 (p = 0.034) 24 112 ± 13 (p = 0.341) 48 111 ±10 (p = 0.256)

The results presented in Table 3 show that were was a statisticallysignificant (p<0.05) increase in Aβ, as represented by Aβ42determination, in the plasma of the mice injected with CG at 5 and 7.5hours after injection. Changes in plasma load of Aβ42 after injectionwith CG were compared to the plasma Aβ level at pretreatment timepoints. As can be observed, there was a significant increase in plasmaAβ levels after injection of CG.

Following a oneweek wash out period, the effect in brain AD level aftercontinuous injection was examined. PS/APP mice were injected every dayfor one week into the periphery, with either CG (number of mice=3) (20mg/kg, ip,) or vehicle (phosphate buffered saline) (number of mice=2),at 11 weeks of age, an age when amyloid pathology in the brain is notvisible. The mice were sacrificed at 12 weeks of age, an age whenamyloid deposition has been initiated and measurable levels of Aβ arepresent in the vehicle treated controls.

The levels of Aβ in the peripheral blood were tested at the end of theadministration period. The levels of Aβ peptides (e.g., Aβ40 or Aβ42) inthe brain and plasma were assessed by ELISA assay. All of the Aβ in thebrain (including Aβ in plaques) was extracted in 70% formic acid (FA).The levels of Aβ peptides in CG treated mice were compared with those ofvehicle treated controls. The results presented in Tables 4 and 5 showthat there was a statistically significant (p<0.05) decrease in Aβ40and/or Aβ42 in the FA-soluble brain fraction in CG-treated mice comparedwith those in control animals tested 1 week after injection. Thiscorrelates with a statistically significant increase in peripheral Aβ40and Aβ42 at the same time point. TABLE 4 Change of plasma Aβ levels 1week following injection with CG Plasma Aβ42 level [% of control]Vehicle 100 ± 9 CG 331 ± 10 treated (p = 0.0081)

TABLE 5 Change of brain Aβ 1week following injection with CG Aβ40[fmol/ml] Aβ42 [fmol/ml] Vehicle 926 ± 26 1209 ± 292 CG 541 ± 50  540 ±271 (P = 0.0031) (P = 0.2038)**Because of data variation, Aβ42 was not statistically significant, p =0.20.

The method of the present invention for determining elevated levels ofAβ in the periphery for the purposes of diagnosing, screening, ormonitoring patient treatment, treatment outcome, or the course and/orseverity of amyloid-related disease in an individual preferably involvesa pretreatment or baseline value for assessing peripheral elevation ofAβ levels in the individual undergoing testing. In the examplespresented herein, an elevation of plasma Abeta was compared bypercentage pre-treatment time point of an individual animal. Similarcomparative assessments of pretreatment and treatment Abeta levels canbe employed for the testing of other mammals, including humans,particularly because the range of Abeta levels can be large between andamong individuals. As a nonlimiting guide, a representative non-elevatedlevel of Abeta in human plasma (e.g., periphery) is about 25%, asdetermined experimentally (e.g., Mehta et al., 2000, Ibid.).

The contents of all patents, patent applications, published PCTapplications and articles, books, references, reference manuals andabstracts cited herein are hereby incorporated by reference in theirentirety to more fully describe the state of the art to which theinvention pertains.

As various changes can be made in the above-described subject matterwithout departing from the scope and spirit of the present invention, itis intended that all subject matter contained in the above description,or defined in the appended claims, be interpreted as descriptive andillustrative of the present invention. Many modifications and variationsof the present invention are possible in light of the above teachings.

1. A method of treating amyloid beta (Aβ)-associated disease, comprisingadministering an amyloid beta (Aβ)-binding agent in the periphery of anindividual in need thereof, wherein said agent binds to Aβ in theperiphery, sequesters Aβ in the periphery and concomitantly decreases Aβlevels in the brain of the individual undergoing treatment, in theabsence of immunomodulating agents.
 2. The method according to claim 1,wherein the amyloid beta (Aβ)-associated disease is selected fromAlzheimer's disease, β-amyloid related problems of Down's syndrome,vascular dementia (cerebral amyloid angiopathy) and amyloidosis.
 3. Themethod according to claim 1, wherein the amyloid beta (Aβ)-binding agentis selected from the group consisting of GM1 ganglioside, gelsolin, anAβ imaging agent, a β-sheet breaker, a β-sheet formation inhibitor and aderivative of an amyloid beta (Aβ)-staining dye.
 4. The method accordingto claim 1, wherein the derivative of the amyloid beta (Aβ)-staining dyeis 1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene and5,5′-{(1,1′biphenyl)-4,4′-diylbis(azo)}bis {2-hydroxybenzoicacid}disodium salt (chrysamine-G).
 5. The method according to claim 1,wherein amyloid beta (Aβ)-binding agent is virtually brain impermeable.6. A method for sequestering Aβ in the periphery comprising blood orblood components of an individual in need thereof, comprising: a)administering an agent having binding affinity for amyloid beta (Aβ) inthe periphery of the individual in need thereof; b) sequestering Aβ inthe periphery, thereby concomitantly decreasing Aβ levels in the brainof the individual.
 7. The method according to claim 6, wherein the agenthaving binding affinity for amyloid beta (Aβ) is administered in theabsence of immunomodulating agents or brain penetrance.
 8. The methodaccording to claim 6, wherein the individual is suffering from anamyloid beta (Aβ)-associated disease.
 9. The method according to claim8, wherein the amyloid beta (Aβ)-associated disease is selected fromAlzheimer's disease, β-amyloid related problems of Down's syndrome,vascular dementia (cerebral amyloid angiopathy) and amyloidosis.
 10. Themethod according to claim 6, wherein the agent having binding affinityfor amyloid beta (Aβ) is selected from the group consisting of GM1ganglioside, gelsolin, an Aβ imaging agent, a β-sheet breaker, a β-sheetformation inhibitor and a derivative of an amyloid beta (Aβ)-stainingdye.
 11. The method according to claim 10, wherein the derivative of theamyloid beta (Aβ)-staining dye is1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene and5,5′-{(1,1′biphenyl)-4,4′-diylbis(azo)}bis {2-hydroxybenzoicacid}disodium salt (chrysamine-G).
 12. A method of monitoring theeffectiveness of drug treatment of beta-amyloid related diseases,comprising: (a) assessing levels of Aβ in the periphery of a recipientof the drug treatment; and (b) determining an elevation of the levels ofAβ in the periphery of the recipient.
 13. The method according to claim12, wherein the levels (Aβ) are assessed by an assay that detects Aβ.14. The method according to claim 13, wherein the assay is (i) aradioisotopic immunoassay or (ii) a non-isotopic immunoassay.
 15. Themethod according to claim 14, wherein the non-isotopic immunoassay isselected from a fluorescent immunoassay, a chemiluminescent immunoassay,or an enzymatic immunoassay (ELISA).
 16. The method according to claim12, wherein the drug treatment comprises an agent having bindingaffinity for amyloid beta (Aβ).
 17. The method according to claim 16,wherein the agent is selected from the group consisting of GM1ganglioside, gelsolin, an Aβ imaging agent, a β-sheet breaker, a β-sheetformation inhibitor and a derivative of an amyloid beta (Aβ)-stainingdye.
 18. The method according to claim 17, wherein the derivative of theamyloid beta (Aβ)-staining dye is1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene and5,5′-{(1,1′biphenyl)-4,4′-diylbis(azo)}bis {2-hydroxybenzoic acid)disodium salt (chrysamine-G).
 19. The method according to claim 12,wherein the monitoring occurs at about 1-25 days followingadministration of drug to the recipient.
 20. The method according toclaim 12, wherein the monitoring occurs at about 5-10 days followingadministration of drug to the recipient.
 21. A method of treatingamyloidosis in a subject, said method comprising administering in theperiphery of said subject an amyloid beta (Aβ) binding agent, said agentselected from the group consisting of GM1 ganglioside, gelsolin, an Aβimaging agent, a β-sheet breaker, a β-sheet formation inhibitor and aderivative of an amyloid beta (Aβ)-staining dye, for a time and underconditions suitable for the agent to bind amyloid beta (Aβ), sequesteramyloid beta (Aβ) in the periphery and decrease amyloid beta (Aβ) levelsin the brain of the individual.
 22. The method according to claim 21,wherein the derivative of the amyloid beta (Aβ)-staining dye is1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene and5,5-′(1,1′biphenyl)-4,4′-diylbis(azo)}bis {2-hydroxybenzoicacid}disodium salt (chrysamine-G).